Unicellular organisms eubacteria archea yeast lecture 27 chapter 21 may 13 2004 jeff esko
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Unicellular Organisms: Eubacteria, Archea, Yeast Lecture 27, Chapter 21 May 13, 2004 Jeff Esko. Overview. General structure of bacterial cell walls Structure, function and assembly of peptidoglycan (murein) Periplasmic  -glucans (MDO) Lipopolysaccharide (LPS) - endotoxin

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Unicellular Organisms: Eubacteria, Archea, Yeast Lecture 27, Chapter 21 May 13, 2004 Jeff Esko

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Unicellular organisms eubacteria archea yeast lecture 27 chapter 21 may 13 2004 jeff esko

Unicellular Organisms: Eubacteria, Archea, Yeast Lecture 27, Chapter 21May 13, 2004Jeff Esko


Overview

Overview

  • General structure of bacterial cell walls

    • Structure, function and assembly of peptidoglycan (murein)

    • Periplasmic -glucans (MDO)

    • Lipopolysaccharide (LPS) - endotoxin

    • Capsular polysaccharides - mimicry

  • Archea glycoproteins - a new area

  • Yeast glycosylation and cell walls


Unicellular organisms eubacteria archea yeast lecture 27 chapter 21 may 13 2004 jeff esko

Capsule

LPS

PG

MDO

Gram-negative bacteria cell wall


Peptidoglycan

A

A

A

A

D

D

D

D

G

G

G

G

A

A

A

A

A

A

A

A

G

G

G

G

D

D

D

D

A

A

A

A

A

A

A

D

D

D

G

G

G

A

A

A

A

A

A

A

G

G

G

G

D

D

D

D

A

A

A

A

A

A

A

A

D

D

D

D

G

G

G

G

A

A

A

A

A

A

A

A

G

G

G

G

D

D

D

D

A

A

A

A

Peptidoglycan

[GlcNAcb1,4MurNAcb1,4]n

Heijenoort (2001) Glycobiology 11:25R


Murein glycopeptide

C

H

O

H

C

H

O

H

2

2

O

O

O

O

O

O

O

H

C

C

H

N

H

A

c

N

H

A

c

3

O

C

L

-

A

l

a

D

-

G

l

u

D

A

P

D

-

A

l

a

D

-

A

l

a

Murein Glycopeptide

Notice D-amino acids

DAP = diaminopimelic acid


Peptidoglycan1

A

A

A

A

D

D

D

D

G

G

G

G

A

A

A

A

A

A

A

A

G

G

G

G

D

D

D

D

A

A

A

A

A

A

A

D

D

D

A

A

A

A

G

G

G

A

A

A

A

A

A

A

G

G

G

G

D

D

D

D

A

A

A

A

A

A

A

A

D

D

D

D

G

G

G

G

A

A

A

A

A

A

A

A

G

G

G

G

D

D

D

D

A

A

A

A

Peptidoglycan

The final step involves cleavage between the D-Ala-D-Ala unit and transpeptidation to the amino group of DAP of another unit


Unicellular organisms eubacteria archea yeast lecture 27 chapter 21 may 13 2004 jeff esko

Vancomycin


Moenomycins

Moenomycins


Peptidoglycan biosynthesis

P

Peptidoglycan Biosynthesis

Undecaprenyl

phosphate


Unicellular organisms eubacteria archea yeast lecture 27 chapter 21 may 13 2004 jeff esko

MDO

Gram-negative bacteria cell wall


Membrane derived oligosaccharides mdo

b2

b2

b2

b2

b2

b2

b2

Membrane Derived Oligosaccharides (MDO)

  • Branched -glucans

  • Represent about 1-5% of dry weight

  • Charged substituents act as an osmolyte and protects the inner membrane against the large difference in osmolarity inside the cell compared to outside the cell

+ Phosphoethanolamine

+ Phosphoglycerol

+ Succinate


Unicellular organisms eubacteria archea yeast lecture 27 chapter 21 may 13 2004 jeff esko

LPS

Gram-negative bacteria cell wall


Lipopolysaccharide lps

Lipopolysaccharide (LPS)

  • LPS consists of three domains

    • Lipid A, otherwise known as endotoxin

    • Core region composed of KDO (K), heptoses (H), and hexoses (open hexagons)

    • Highly variable outer O-antigen region


Unicellular organisms eubacteria archea yeast lecture 27 chapter 21 may 13 2004 jeff esko

H

O

O

H

H

O

H

O

H

O

U

r

i

d

i

n

e

U

r

i

d

i

n

e

N

H

AcN

O

H

O

H

O

H

O

H

H

O

H

O

O

O

O

O

O

O

O

P

P

P

P

O

O

O

C

O

O

O

O

O

  • Initiates by acylation of UDP-GlcNAc at C3, followed by N-deacetylation, and N-acylation

Wyckoff et al. (1998) Trends Microbiol. 6:154


Lipid a assembly

O

H

O

H

O

H

C

H

2

O

O

O

O

H

O

O

O

H

O

H

O

O

O

O

O

O

H

O

O

U

r

i

d

i

n

e

N

H

P

P

N

H

N

H

O

P

O

C

O

O

O

C

O

O

C

O

O

N

H

P

O

O

H

O

H

O

O

O

C

O

O

H

O

O

O

H

H

O

H

O

H

O

H

O

H

O

H

O

H

O

H

O

Lipid A Assembly

+

Diacylglucosamine-1-P condenses with another molecule of UDP-diacylglucosamine to form the tetraacyl disaccharide core


Lipid a assembly1

Lipid A Assembly

  • KDO transferases initiate the formation of the core

  • Additional C12 fatty acids added to -hydroxy groups (wax)

  • Lipid A translocates to the outer leaflet of the outer membrane by msbA (ABC transporter)

Doerrler et al. (2001) J Biol Chem. 276:11461


Lipid a biology

Lipid A Biology

  • Lipid A, the heat stable endotoxin of gram negatives

  • Resistant strains of mice defined a locus, lps, which was positionally cloned.

  • lps turned out to be homologous to toll receptors in Drosophila, which were known to be involved in innate immunity to fungal infection

  • lps turns out to beTlr4, a member of a family of signaling receptors (10 members known).

    • Tlr4 binds to Lipid A.

    • Tlr2 apparently binds and responds to muramyl-dipeptide


Unicellular organisms eubacteria archea yeast lecture 27 chapter 21 may 13 2004 jeff esko

Takeda & Akira (2001)Genes to Cells 6:733


Lps structural and functional domains

LPS Structural and Functional Domains


Core region contains unusual sugars

C

H

O

H

2

C

H

O

H

2

H

O

H

C

C

H

O

H

O

C

O

O

H

O

H

O

O

H

O

H

O

H

O

H

O

H

O

H

Core region contains unusual sugars

  • The inner core contains 1-4 KDO residues, which look like an analog of sialic acid.

  • The core also contains heptopyranoses, which can vary stereochemically

  • The rest of the core consists of various combinations of Glc and Gal

3-deoxy-D-manno-octulosonic acid (KDO)

L-glycero-D-manno-

heptulose


Lps structural and functional domains1

LPS Structural and Functional Domains


O antigens

O-antigen

Structure

O9

a

3

a

2

a

2

a

2

a

3

O6

a

3

b

4

b

3

a

4

b

2

O124

b

3

b

6

a

3

a

4

G

l

c

L

A

b

6

GlcLA = glucolactillic acid

O-antigens

  • O-antigens consist of 2-8 sugars, repeated ≤50 times

  • O-antigens gives rise to different serotypes and some are correlated with disease


Unicellular organisms eubacteria archea yeast lecture 27 chapter 21 may 13 2004 jeff esko

Capsule

Gram-negative bacteria cell wall


Unicellular organisms eubacteria archea yeast lecture 27 chapter 21 may 13 2004 jeff esko

a4

b4

a4

b4

a4

a8

a8

a8

a8

a8

b4

b3

b4

b3

b4

Capsule type

Structure

K1, polysialic acid

K5, N-acetylheparosan

Group A Streptococcus

(hyaluronan)

  • Mucoid strains contain a polysaccharide capsule

  • >80 different capsules types are known just in E. coli

  • Extraordinary diversity of structure


Mycobacteria

Mycobacteria

Crick et al. (2001) Glycobiology 11:107R


Unicellular organisms eubacteria archea yeast lecture 27 chapter 21 may 13 2004 jeff esko

Crick et al. (2001) Glycobiology 11:107R


Unicellular organisms eubacteria archea yeast lecture 27 chapter 21 may 13 2004 jeff esko

Crick et al. (2001) Glycobiology 11:107R


Bacterial glycoproteins

Bacterial Glycoproteins

  • Surface-layer (S-layer) glycoproteins

  • Prevalent in Bacteria and Archaea, but structures differ, e.g., N-linked glycosylation only in Archaea

  • Structural analyses have revealed unusual carbohydrate-linkage regions….

  • ….and unusual nucleoside diphosphate-linked oligosaccharides


Unicellular organisms eubacteria archea yeast lecture 27 chapter 21 may 13 2004 jeff esko

Schaffer et al (2001) Proteomics 1:248


Unicellular organisms eubacteria archea yeast lecture 27 chapter 21 may 13 2004 jeff esko

Notice diversity of linkages

Similarity in sequence of N-linked attachment sites and mechanism of assembly of glycan (dolichol pathway)

Burda & Aebi (1999) Biochim Biophys Acta 1426:239


Yeast make asn linked glycoproteins

Yeast make AsN-linked Glycoproteins

  • Yeast make membrane N-linked glycoproteins much in the same way as higher eukaryotes

  • Serves as a model for human genetic diseases (CDG)

M Aebi (2001) Trends in Cell Biology 11:136


Yeast mannans

a 6

a 6

a 2

a 3

a 2

a 6

a 2

a 3

a 2

a 6

a 2

a 3

a 2

a 3

a 6

a 2

a 3

a 2

a 3

a 3

a 2

a 6

a 6

a 3

-1-P

a 2

a 4

a 3

a 6

a 6

-P-

a 2

a 3

a 2

a 2

a 3

a 6

a 2

a

b4

a

Ser/Thr

Ser/Thr

b4

Vertebrate

a-dystroglycan

Asn

Yeast Mannans

Willer et al (2003) Curr Opin Struct Biol 13:621


Yeast cell walls

GPI

proteins

GPI

proteins

GPI

proteins

Pir cell wall

proteins

b1,6-glucans

b1,6-glucans

b1,6-glucans

b1,3-glucans

Plasma

membrane

Yeast Cell Walls

The cell wall is made of 60% b-glucans, 40% mannoproteins, and ~1% chitin

Smits et al (2001) Microbiology 147:781


Summary

Summary

  • Bacterial cell walls are complex layered structures composed of multiple classes of glycans

  • Bacterial wall glycans provide an exoskeleton, define shape, protect against turgor pressure, create antigenic serotypes, and provide a way to prevent immune recognition

  • Glycosylated proteins are present in eubacteria and in archaea

  • Yeast produce many of the same glycans found in higher eukaryotes, but the mecahnism of assembly differs in subtle way

  • Yeast cell walls are composed of glycans (glucans and chitin)

  • Yeast and bacteria provide powerful genetic systems for understanding membrane and cell wall assembly


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